In general, particle processing (e.g., cytometry) systems (e.g., cytometers) and methods are known. For example, some approaches to particle processing or analyzing (e.g., cell purification) systems such as sorting flow cytometers and other particle processing systems have proven to be useful in life science research, industrial, agricultural, diagnostics, and other medical applications.
In general, a cytometer can be described as a system that can measure large numbers of homogeneous and/or heterogeneous particle sets to achieve statistically relevant data sets that can be used to group and/or identify subpopulations that reside within a given particle population (e.g., within one or more samples). These measurements are sometimes performed optically (whether they are intrinsic or responsive to an optical stimulus), or they may be electrical in nature (or some other physical, chemical, or biological characteristic) as a stream of particles passes through a measurement or inspection zone. The particle sets may include biological entities such as cells (e.g., bacteria, viruses, organelles, yeasts, spores, genetic material, spermatozoa, egg cells, multicellular organisms), or other organisms, or other naturally occurring or synthetic/synthetically derived objects.
With the addition of sort functionality, a cytometer can also be used to isolate (e.g., physically separate) one or more particles of interest from a given sample through operator control. See, e.g., U.S. Pat. No. 6,248,590, the entire content of which is hereby incorporated by reference in its entirety. In general, this technique can be used to classify and/or separate (e.g., purify or enrich) one or more populations as defined by the operator.
Cell purification means, such as flow cytometry, can be used to process microscopic particles of biological interest, such as cells or viruses, based on optical properties of the particles. However, when multiple sensors are in use, there exists the possibility that attributive interference or optical crosstalk may occur, which can limit the ability to provide broad accurate dynamic measurement ranges for the sensing locations and/or particles of interest.
For example, it is desired that any light emanating from one particle sensing location should not interfere with the light being measured from another particle sensing location. If there is any such optical crosstalk interference, then some measurements made may be erroneous, and the further data analysis and/or further processing steps (such as producing a diagnostic assessment, or separation based on measured and/or differentiated characteristics) are likely to be affected.
Some measured characteristics of particles positioned at one sensing location may then mask or be masked by characteristics of another particle at another location. An example of this effect may be seen when a very low-response (e.g. dimly fluorescent) particle is to be measured while a particle with bright fluorescence happens to be within an additional sensing location within a similar timeframe. As a non-limiting example, if there is potential for optical crosstalk within a given system, the dim particle may be erroneously measured as being brighter than it actually is, since an amount of light signal emanating from the bright particle may also be captured.
The likelihood of such optical crosstalk may be increased when there is close proximity of sensing locations, or when particles are located on the same substrate or sensing region, or other common optical componentry or light paths. Thus, measurement accuracy may be compromised, which could cause issues in diagnostic applications, e.g., where critical treatment decisions are based on such measurements.
Additionally, in cell purification applications and cell sorting, such erroneous measurements may restrict the ability to provide suitable sub-populations with suitable purity, recovery, and/or yield, since unwanted particles or cells may be inadvertently separated based on inaccurate particle classification. It is therefore desirable to have systems and methods for reducing such optical crosstalk in particle analysis systems and/or cell purification systems.